Hiroshi Wakao

Mammary gland factor (MGF) is a novel member of the cytokine regulated transcription factor gene family and confers the prolactin response.

Milk protein gene expression in mammary epithelial cells is regulated by the action of the lactogenic hormones insulin, glucocorticoids and prolactin. The mammary gland factor, MGF, has been shown to be a central mediator in the lactogenic hormone response. The DNA binding activity of MGF is hormonally regulated and essential for beta‐casein promoter activity. We have used Red A Sepharose‐ and sequence‐specific DNA affinity chromatography to purify MGF from mammary gland tissue of lactating sheep. Proteins of 84 and 92 kDa were obtained, proteolytically digested and the resulting peptides separated by reverse phase high pressure liquid chromatography. The 84 and 92 kDa proteins yielded very similar peptide patterns. The amino acid sequence of two peptides was determined. The sequence information was used to derive oligonucleotide probes. A cDNA library from the mRNA of mammary gland tissue of lactating sheep was screened and a molecular clone encoding MGF was isolated. MGF consists of 734 amino acids and has sequence homology with the 113 (Stat113) and 91 kDa (Stat91) components of ISGF3, transcription factors which are signal transducers of IFN‐alpha/beta and IFN‐gamma. Two species of MGF mRNA of 6.5 and 4.5 kb were detected in mammary gland tissue of lactating sheep. Lower mRNA expression was found in ovary, thymus, spleen, kidney, lung, muscle and the adrenal gland. MGF cDNA was incorporated into a eukaryotic expression vector and cotransfected with a vector encoding the long form of the prolactin receptor into COS cells. A strong MGF‐specific bandshift was obtained with nuclear extracts of COS cells induced with prolactin. Treatment of activated MGF with a tyrosine‐specific protein phosphatase resulted in the loss of DNA binding activity. Prolactin‐dependent transactivation of a beta‐casein promoter‐luciferase reporter gene construct was observed in transfected cells.

Interleukin-3, granulocyte-macrophage colony stimulating factor and interleukin-5 transduce signals through two STAT5 homologs.

Interleukin‐3 (IL‐3) is an important regulator of hemopoiesis and considerable effort has been directed towards the study of its mechanism of signal transduction. In this paper, we describe the first molecular identification of a STAT transcription factor that is activated by IL‐3. STATs exist in a cytoplasmic, transcriptionally inactive form which, in response to extracellular signals, become tyrosine phosphorylated and translocate to the nucleus where they bind to specific DNA elements. Several of these DNA elements were found which bind proteins in an IL‐3‐responsive manner. Analysis of these bandshift complexes with available antibodies to the known STATs suggests that IL‐3 activates the DNA‐binding ability of STAT5, a protein which was originally characterized as a prolactin‐responsive transcription factor in sheep. IL‐5 and granulocyte‐macrophage colony stimulating factor (GM‐CSF), which share a common signaling receptor subunit with IL‐3, also activate STAT5. Unexpectedly, two murine STAT5 homologs, 96% identical to each other at the amino acid level, were isolated and IL‐3‐dependent GAS binding could be reconstituted in COS cells transfected with IL‐3 receptor and either STAT5 cDNA. In IL‐3‐dependent hemopoietic cells, both forms of STAT5 are expressed and activated in response to IL‐3.

Prolactin induces phosphorylation of Tyr694 of Stat5 (MGF), a prerequisite for DNA binding and induction of transcription.

Mammary gland factor (MGF) is a transcription factor discovered initially in the mammary epithelial cells of lactating animals. It confers the lactogenic hormone response to the milk protein genes. We reported recently the isolation of the cDNA encoding MGF. MGF is a novel member of the cytokine‐regulated transcription factor gene family. Members of this gene family mediate interferon alpha/beta and interferon gamma induction of gene transcription, as well as the response to epidermal growth factor and interleukin‐6, and have been named signal transducers and activators of transcription (Stat). The name Stat5 has been assigned to MGF. We studied the mechanisms involved in the prolactin activation of Stat5 in COS cells co‐transfected with cDNA encoding Stat5 and the prolactin receptor. Prolactin treatment of the transfected cells caused activation of Stat5 within 5‐10 min. This activation does not require ongoing protein synthesis. Tyrosine kinase inhibitors prevent Stat5 activation in transfected COS cells. Treatment of recombinant Stat5 with a tyrosine‐specific protein phosphatase in vitro abolishes its DNA binding activity. Prolactin stimulation of transfected cells induces Stat5 phosphorylation on tyrosine. Phosphorylation of in vitro transcribed and translated Stat5 with the Jak2 tyrosine kinase, but not with fyn, lyn or lck, confers DNA binding activity. The prolactin response of the beta‐casein milk protein gene promoter can be observed in COS cells transfected with cDNA vectors encoding Stat5 and the long form of the prolactin receptor. The short form of the prolactin receptor is unable to promote Stat5 phosphorylation and confer transcriptional induction in COS cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Suppression of interleukin-3-induced gene expression by a C-terminal truncated Stat5: role of Stat5 in proliferation.

Interleukin‐3 (IL3) was shown recently to utilize the transcription factor Stat5, but the genes regulated by this pathway and the biological consequence of Stat5 activation remained to be determined. In order to study the role of Stat5 in IL3 signalling, we constructed a dominant‐negative Stat5 protein by C‐terminal truncation, and inducibly expressed it in an IL3‐dependent cell line. The effect of dominant‐negative Stat5 induction on expression of IL3 early response genes was examined, and expression of several genes, including cis, osm and pim‐1 was inhibited profoundly. The expression of c‐fos was also reduced, but to a lesser extent. While activated Ras alone (though not Stat5 alone) could induce c‐fos, maximal expression required the action of both Ras and Stat5. Interestingly, although the membrane‐proximal region of the IL3 receptor beta‐chain is responsible for both Jak2‐Stat5 activation and c‐myc induction, c‐myc levels were not affected by the dominant‐negative Stat5. Thus, the signals directed by this membrane‐proximal domain, which is essential for transducing a DNA synthesis signal, can be separated further into Stat5 or c‐myc pathways. The net effect of dominant‐negative Stat5 expression was partial inhibition of IL3‐dependent growth. This provides the first direct evidence that Stat5 is involved in regulation of cell proliferation.

Prolactin, growth hormone, erythropoietin and granulocyte-macrophage colony stimulating factor induce MGF-Stat5 DNA binding activity.

The molecular components which mediate cytokine signaling from the cell membrane to the nucleus were studied. Upon the interaction of cytokines with their receptors, members of the janus kinase (Jak) family of cytoplasmic protein tyrosine kinases and of the signal transducers and activators of transcription (Stat) family of transcription factors are activated through tyrosine phosphorylation. It has been suggested that the Stat proteins are substrates of the Jak protein tyrosine kinases. MGF‐Stat5 is a member of the Stat family which has been found to confer the prolactin response. MGF‐Stat5 can be phosphorylated and activated in its DNA binding activity by Jak2. The activation of MGF‐Stat5 is not restricted to prolactin. Erythropoietin (EPO) and growth hormone (GH) stimulate the DNA binding activity of MGF‐Stat5 in COS cells transfected with vectors encoding EPO receptor and MGF‐Stat5 or vectors encoding GH receptor and MGF‐Stat5. The activation of DNA binding by prolactin, EPO and GH requires the phosphorylation of tyrosine residue 694 of MGF‐Stat5. The transcriptional induction of a beta‐casein promoter luciferase construct in transiently transfected COS cells is specific for the prolactin activation of MGF‐Stat5; it is not observed in EPO‐ and GH‐treated cells. In the UT7 human hematopoietic cell line, EPO and granulocyte‐macrophage colony stimulating factor activate the DNA binding activity of a factor closely related to MGF‐Stat5 with respect to its immunological reactivity, DNA binding specificity and molecular weight. These results suggest that MGF‐Stat5 regulates physiological processes in mammary epithelial cells, as well as in hematopoietic cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Transcriptional regulation of the cyclin D1 promoter by STAT5: its involvement in cytokine‐dependent growth of hematopoietic cells

STAT5 is a member of a family of transcription factors that participate in the signal transduction pathways of many hormones and cytokines. Although STAT5 is suggested to play a crucial role in the biological effects of cytokines, its downstream target(s) associated with cell growth control is largely unknown. In a human interleukin‐3 (IL‐3)‐dependent cell line F‐36P‐mpl, the induced expression of dominant‐negative (dn)‐STAT5 and of dn‐ras led to inhibition of IL‐3‐dependent cell growth, accompanying the reduced expression of cyclin D1 mRNA. Also, both constitutively active forms of STAT5A (1*6‐STAT5A) and ras (H‐rasG12V) enabled F‐36P‐mpl cells to proliferate without added growth factors. In NIH 3T3 cells, 1*6‐STAT5A and H‐rasG12V individually and cooperatively transactivated the cyclin D1 promoter in luciferase assays. Both dn‐STAT5 and dn‐ras suppressed IL‐3‐induced cyclin D1 promoter activities in F‐36P‐mpl cells. Using a series of mutant cyclin D1 promoters, 1*6‐STAT5A was found to transactivate the cyclin D1 promoter through the potential STAT‐binding sequence at −481 bp. In electrophoretic mobility shift assays, STAT5 bound to the element in response to IL‐3. Furthermore, the inhibitory effect of dn‐STAT5 on IL‐3‐dependent growth was restored by expression of cyclin D1. Thus STAT5, in addition to ras signaling, appears to mediate transcriptional regulation of cyclin D1, thereby contributing to cytokine‐dependent growth of hematopoietic cells.

Tyrosine 343 in the erythropoietin receptor positively regulates erythropoietin-induced cell proliferation and Stat5 activation.

While previous studies with truncated erythropoietin receptors (EpRs) have suggested that the tyrosine phosphorylation of the EpR does not play a role in Ep‐induced proliferation, we have found, using a more subtle, full length EpR mutant, designated Null, in which all eight of the intracellular tyrosines have been substituted with phenylalanine residues, that Null cells require substantially more Ep than wild‐type cells in order to proliferate as efficiently. A comparison of Ep‐induced proliferation with Ep‐induced tyrosine phosphorylation patterns, using wild‐type and Null EpR‐expressing cells, revealed that Stat5 tyrosine phosphorylation and activation correlated directly with proliferation. Moreover, studies with a Y343F EpR point mutant and various EpR deletion mutants revealed that both Ep‐induced proliferation and Stat5 activation were mediated primarily through Y343, but that other tyrosines within the EpR could activate Stat5 in its absence.

Interleukin 2 and erythropoietin activate STAT5/MGF via distinct pathways.

Signal transducers and activators of transcription (STAT) proteins play an important role in cytokine signal transduction in conjunction with Janus kinases (JAKs). MGF/STAT5 is known as prolactin regulated STAT. Here we demonstrate that interleukin 2 (IL‐2) as well as erythropoietin (EPO) stimulate STAT5 and induce tyrosine phosphorylation of STAT5. These IL‐2‐ and EPO‐induced STATs have an identical DNA binding specificity and immunoreactivity. We also show that IL‐4 induces a DNA binding factor which possesses similar, but distinct, DNA binding specificity from that of STAT5 and is immunologically different from STAT5. Analysis of two EPO receptor (EPOR) transfected CTLL‐2 cell lines discloses that IL‐2 activates JAK1 and JAK3 as well as STAT5, while EPO stimulates STAT5 and JAK2 in EPO‐responsive CTLL‐2 cells (ERT/E2). On the contrary, EPO activates neither JAK2 nor STAT5 in other cell lines that failed to respond to EPO (ERT cells). EPOR and JAK2 associate with each other regardless of EPO presence in ERT/E2 cells, however, such an interaction is not present in ERT cells. Thus, EPOR and JAK2 association seems to be important for EPO responsiveness in CTLL‐2 cells.

Generation of cloned mice by direct nuclear transfer from natural killer T cells

Cloning mammals by nuclear transfer (NT) remains inefficient. One fundamental question is whether clones have really been derived from differentiated cells rather than from rare stem cells present in donor-cell samples. To date, cells, such as mature lymphocytes, with genetic differentiation markers have been cloned to generate mice only via a two-step NT involving embryonic stem (ES) cell generation and tetraploid complementation [1, 2 and 3]. Here, we show that the genome of a unique T-cell population, natural killer T (NKT) cells, can be fully reprogrammed by a single-step NT. The pups and their placentas possessed the rearranged TCR loci specific for NKT cells. The NKT-cell-cloned embryos had a high developmental potential in vitro: Most (71%) developed to the morula/blastocyst stage, in marked contrast to embryos from peripheral blood T cells (12%; p < 1 x 10(-25)). Furthermore, ES cell lines were efficiently established from these NKT-cell blastocysts. These findings clearly indicate a high level of plasticity in the NKT-cell genome. Thus, differentiation of the genome is not always a barrier to NT cloning for either reproductive or therapeutic purposes, so we can now postulate that at least some mammals cloned to date have indeed been derived from differentiated donor cells.

A common polymorphism in the interleukin 8 gene promoter is associated with Clostridium difficile diarrhea.

In mouse liver transplantation, tolerance is readily inducible. Recent studies have revealed that CD25+CD4+ regulatory T cells play an important role in regulating various immune responses, including transplant tolerance. However, the contribution of these cells to tolerance in mouse liver transplantation has not been elucidated. We showed here that depletion of CD25+CD4+ T cells increased proliferative response of CD4+ T cells and cytotoxic T lymphocyte induction of CD8+ T cells. Depletion of these cells in the recipient but not in the donor before liver transplantation caused rejection. Furthermore, the number of CD25+CD4+ population and forkhead/winged helix transcription factor expression in liver mononuclear lymphocytes derived from tolerant mice were higher than those from grafts undergoing rejection. In conclusion, these results indicate that CD25+CD4+ regulatory T cells in the recipient but not in the donor of liver transplantation are important for the tolerance induction. Liver Transpl 12:1112–1118, 2006.